Project Summary/Abstract The overall goal of this proposal is to enhance the efficacy of CDK4/6 inhibitors for melanoma treatment, and to identify novel, highly effective combinatorial treatments for melanoma patients involving CDK4/6 inhibitors. Cyclin dependent kinases CDK4 and CDK6 are components of the core cell cycle machinery that are activated upon interaction with their regulatory subunits, the D-type cyclins (cyclins D1, D2 and D3). Chemical inhibitors of the CDK4/6 kinase are currently being used in over 240 clinical trials for different cancer types, including melanomas. A large number of studies demonstrated that treatment of human cancer cells with CDK4/6 inhibitors blocks tumor cell proliferation, and in some cases causes tumor cell senescence. In our preliminary studies, we observed that tumor cell lines expressing high levels of cyclin D3-CDK6 complexes undergo cell death upon CDK4/6 inhibition. We found that cyclin D3-CDK6, but not several other types of cyclin D-CDK4/6 complexes (such as cyclin D1-CDK4), plays a rate-limiting role in regulating tumor cell metabolism, and protects tumor cells against elevated levels of reactive oxygen species. In another study, we found that in mouse and human tumor cells, cyclin D-CDK4 kinase regulates the levels of an immune checkpoint protein, programmed death-ligand 1 (PD-L1). Using mouse cancer models and in vitro cultured cells, we found that treatment with CDK4/6 inhibitors results in a strong upregulation of PD-L1 protein levels in tumor cells. We also found that combined administration of CDK4/6 inhibitors with antibodies that target the interaction between programmed cell death protein 1 (PD-1) and its ligand PD-L1 increased the efficacy of immune checkpoint therapy. In the studies proposed here, we will test novel therapeutic approaches involving CDK4/6 inhibition in vivo for treatment of melanomas. In Aim 1, we will test our hypothesis that measuring cyclin D3 and CDK6 levels in metastatic melanomas may allow one to identify tumor cases that are particularly amenable to CDK4/6 inhibitor treatment, as they would undergo death of tumor cells (and of melanoma metastatic cells) upon CDK4/6 inhibition. We will also explore ways to trigger killing of melanoma cells by CDK4/6 inhibitors, in case of melanomas not expressing high cyclin D3/CDK6 levels. We will address these issues using a very large and unique collection of melanoma patient-derived xenografts, and short-term cultures of primary human metastatic melanoma cells. In Aim 2, we will test our hypothesis that CDK4/6 inhibitor treatment would increase the efficacy of immune checkpoint therapy for melanomas, and result in tumor regression and improved survival rates in mouse melanoma models. We will also study how treatment of melanoma-bearing mice with CDK4/6 inhibitors affects the anti-tumor immune response at the organismal level. The expected overall impact of this proposal is that it will identify a subset of melanomas that are particularly sensitive to anti-CDK4/6 therapy, and identify novel, highly effective combinatorial treatments for melanoma patients involving CDK4/6 inhibitors.